The nuclear organization of the DNA is an important aspect of the expression of eukarvotic genes, but little is known about the mechanisms regulating the three-dimensional arrangement of the chromatin fiber within the nucleus of interphase cells. Chromatin insulators or boundary elements appear to be important players in the establishment of this arrangement, as they are thought to regulate the expression of eukaryotic genes by establishing higher order domains of chromatin organization. The gypsy insulator is perhaps the best-studied example of one of these sequences. It is composed of 12 DNA binding sites that interact with the suppressor of Hairy-wing [su(Hw)] protein. A second component of the gypsy insulator, the mod(mdg4) protein, does not bind to DNA directly and its presence in the insulator is due to its direct interaction with su(Hw). The components of the insulator are present at several hundred sites on polytene chromosomes, but in interphase nuclei the proteins concentrate at 10-20 sites located mostly in the nuclear lamina. This localization is disrupted by mutations in trithorax-and Polycomb- group genes, suggesting an involvement of these proteins in insulator function. These results suggest the components of the gypsy insualtor and trxG and PcG proteins participate in the same cellular processes by playing a major role in the organization of the chromatin fiber within the nucleus. In this application, we propose to continue investigating the mechanisms of insulator function by identifying and isolating additional protein components of the gypsy insulator. These proteins will be characterized and their contribution to various aspects of insulator function will be studied in detail. The mechanisms by which the insulator affects gene expression will be studied by analyzing its effect on the structure and organization of the chromatin fiber. Effects on higher order chromatin structure will be analyzed by determining the role of histone acetylation on insulator function. The role of the insulator in the subnuclear organization of the DNA will be analyzed using su(Hw)- GFP fusion proteins and determining the pattern and distribution of insulator sites in interphase nuclei; changes in this pattern during cell differentiation and induction of gene expression will be analyzed. Finally, changes in the higher order structure of chromatin will be studied by electron microscopy. From these studies we hope to gain insights into the organization of the DNA in the nucleus and its role in the control of gene expression in eukaryotes.